Vaults are large cytoplasmic ribonucleoprotein particles that possess a distinct barrel- shaped morphology, have a mass of 13 million daltons, and are composed of four proteins and a small RNA. Vaults are found in most, if not all, eukaryotic organisms including the lower eukaryote Dictyostelium discoideum. Although vaults are largely cytoplasmic, a subpopulation of vaults has been localized to the nuclear membrane at nuclear pore complexes. The cDNAs for the two major Dictyostelium vault proteins (MVPalpha and MVPbeta) have been isolated. The MVPalpha cDNA has been used to disrupt the MVPalpha gene in Dictyostelium resulting in a cell line with altered vault structure. The MVPbeta cDNA will be used to delete the MVPbeta gene by double homologous recombination in order to produce cell lines devoid of the MVPbeta protein. Double MVPalpha/MVPbeta disruptants will also be isolated. Dr. Rome will examine important features of cellular metabolism and physiology in these mutant lines to determine the cellular functions in which vault play a role. Cloning of a cDNA for the rat major vault protein has allowed the extension of these studies to higher eukaryotes. Stably transformed rat and mouse lines expressing antisense mRNA have been isolated. These cells will be examined for functional consequences of a reduced level of vault expression. Dr. Rome designed a strategy for identifying a yeast major vault protein. Once identified, the yeast MVP gene will be cloned, sequenced, and used to determine if yeast contain vaults and if these structures are essential to yeast growth and survival.